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Contents

Overview

Cryopreserving PBMC allows for phenotyping or functional studies to be preformed at a future time for convenience or efficiency, but cryopreservation adversely affects the survival and function of leukocyte populations differentially and the user should make themselves aware of the literature on this.

This protocol has been adapted from the URECA study of the Inner City Asthma Consortium

Procedure

This is a sterile procedure. Perform all steps in a hood (biological safety cabinet), observing universal precautions for blood specimens and all biosafety regulations. All reagents should be used at room temperature.

Take Freezing Media A and B out of the freezer. For each 10 x 106 cells, 0.5 mL of A and B will be needed. Allow time for the media to reach room temperature.

After isolating and counting PBMC as per your protocol, pellet cells to be preserved one additional time, remove supernatant and loosen pellet for resuspension in freezing media.

Calculate the volume of PBS containing 1% Freezing Media A to slowly add (drop by drop to the side of tube to minimize shearing forces) to the cells to yield a concentration of 20 x 106 cells/mL. Use a sterile pipette to add PBS containing 1% Freezing Media A to the cells to adjust the concentration to 20 x 106 cells/mL. Mix gently.

Add an equal amount of Freezing Media B drop by drop while turning the tube. This should take 2 minutes. The final cell concentration will now be 10 x 106 cells/mL.

Once mixing is complete, prepare the PBMC aliquots which will be frozen. Pipette gently to minimize sheer forces!

Aliquot to cryovials (10-20 million per tube)

Place the cryovials in a room temperature Nalgene freezing container with 2-propanol. Place the container in the freezer (– 80 °C) for at least 12 hours. After 12 hrs, the cryovials can be transferred to liquid nitrogen for long-term storage.